Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system (CNS) affecting primarily young adults in their most productive age, and therefore causing a significant disability. While its etiology remains elusive, most evidence supports the autoimmune pathogenesis of the disease. According to this hypothesis, the activation of autoreactive T-cells is a central event in the development of autoimmune response in MS. The objective of this proposal is to examine molecular events involved in the initiation of autoimmune response in MS. Recent studies have reported an unexpectedly high degree of T-cell receptor (TCR) degeneracy and molecular mimicry as a frequent phenomenon that might play a role in the initiation of autoimmune response in MS. MHC DR2-biased combinatorial peptide libraries are developed as a tool to characterize degenerate T-cells. As auto-antigens are predominantly weak TCR ligands, we propose that myelin-reactive T-cells may be over-represented among the cells with a degenerate TCR. Myelin basic protein (MBP)-specific T-cells exhibit decreased CD28 co-stimulatory requirements in MS patients when compared to healthy controls. We hypothesize that dysregulation of co-stimulatory pathways play a role in the initial activation, prolonged survival, and the expansion of autoreactive cells in MS. Co-stimulation-independent activation might be particularly relevant for the autoantigen recognition within the CNS, as local inflammatory environment enhances autoantigen presentation even in the absence of co-stimulatory molecules on the resident antigen presenting cells. We plan to achieve our objective by pursuing the following Specific Aims: 1) Determine the frequency and TCR repertoire of T-cell clonotypes with a high degree of TCR flexibility. 2) Define co-stimulation requirements for the activation and growth characteristics of T-cells with flexible TCR in RR MS patients and OND controls. 3) Identify mechanisms by which the inflammatory environment induces the autoreactive T-cell activation. The information provided by these studies may yield important insights into the physiologic and pathologic role of the autoreactive T cells, and characterize structurally and functionally the specific targets for the new therapies of MS. [unreadable] [unreadable]